Extracellular Vesicles in Inflammatory Bowel Disease: Small Particles, Big Players.

Extracellular vesicles are nanovesicles released by many cell types into the extracellular space. They are important mediators of intercellular communication, enabling the functional transfer of molecules from one cell to another. Moreover, their molecular composition reflects the physiological status of the producing cell and tissue. Consequently, these vesicles have been involved in many [patho]physiological processes such as immunomodulation and intestinal epithelial repair, both key processes involved in inflammatory bowel disease. Given that these vesicles are present in many body fluids, they also provide opportunities for diagnostic, prognostic, and therapeutic applications. In this review, we summarise functional roles of extracellular vesicles in health and disease, with a focus on immune regulation and intestinal barrier integrity, and review recent studies on extracellular vesicles and inflammatory bowel disease. We also elaborate on their clinical potential in inflammatory bowel disease.

Roles for Gcn5p and Ada2p in transcription and nucleotide excision repair at the Saccharomyces cerevisiae MET16 gene.

Chromatin structure, transcription and repair of cyclobutane pyrimidine dimers at the MET16 gene of wild type, gcn5Delta and ada2Delta Saccharomyces cerevisiae cells were studied under repressing or derepressing conditions. These two components of the SAGA/ADA chromatin remodelling complexes are expendable for the basal transcription of MET16 but are mandatory for its full transcription induction. Despite their influence on transcription neither protein induces major changes in MET16 chromatin structure, but some minor ones occur. Repair at the coding region of the transcribed strand is faster than repair at non-transcribed regions in all strains and either growth condition. Moreover, the more MET16 is transcribed the faster the repair. The data show that by changing the transcription extent the rate of repair at each DNA strand is altered in a different way, confirming that repair at this locus is strongly modulated by its chromatin structure and transcription level. Deletion of GCN5 or ADA2 reduces repair at MET16. The results are discussed in light of the current understanding of Gcn5p and Ada2p functions, and they are the first to report a role for Ada2p in the nucleotide excision repair of the regulatory and transcribed regions of a gene.

Cbf1p modulates chromatin structure, transcription and repair at the Saccharomyces cerevisiae MET16 locus.

The presence of damage in the transcribed strand (TS) of active genes and its position in relation to nucleosomes influence nucleotide excision repair (NER) efficiency. We examined chromatin structure, transcription and repair at the MET16 gene of wild-type and cbf1Delta Saccharomyces cerevisiae cells under repressing or derepressing conditions. Cbf1p is a sequence-specific DNA binding protein required for MET16 chromatin remodelling. Irrespective of the level of transcription, repair at the MspI restriction fragment of MET16 exhibits periodicity in line with nucleosome positions in both strands of the regulatory region and the non-transcribed strand of the coding region. However, repair in the coding region of the TS is always faster, but exhibits periodicity only when MET16 is repressed. In general, absence of Cbf1p decreased repair in the sequences examined, although the effects were more dramatic in the Cbf1p remodelled area, with repair being reduced to the lowest levels within the nucleosome cores of this region. Our results indicate that repair at the promoter and coding regions of this lowly transcribed gene are dependent on both chromatin structure and the level of transcription. The data are discussed in light of current models relating NER and chromatin structure.

Distinction of basaloid squamous cell carcinoma from adenoid cystic and small cell undifferentiated carcinoma by immunohistochemistry.

Basaloid squamous cell carcinoma (BSCC) is a recently recognized variant of squamous cell carcinoma (SCC) with a predilection to occur in the tongue base, hypopharynx, and supraglottic larynx. In smal biopsy specimens, these tumors can be difficult to distinguish from small cell undifferentiated carcinoma (SCUC) and adenoid cystic carcinoma (ACC). Monoclonal antibodies reactive with cytokeratin (AE1/AE3, 34betaE12, Cam 5.2) as well as a variety of other cellular antigens (vimentin, actin, desmin, chromogranin, synaptophysin, CD57, neuron-specific enolase [NSE], and S100) were used in an immunoperoxidase method with paraffin-embedded tissue to phenotypically characterize 23 cases of BSCC, 10 cases of SCUC, and 15 cases of ACC. The neoplastic cells in 22 of the 23 cases of BSCC reacted with the high-molecular-weight cytokeratin antibody 34betaE12, whereas no reactivity was seen in any of the 10 cases of SCUC. This pattern of 34betaE12 reactivity more consistently differentiated BSCC from SCUC than did reactivity with the neuroendocrine markers chromogranin, synaptophysin, CD57, and NSE. These findings show that immunoperoxidase stains performed on paraffin-embedded tissue are potentially useful in establishing a diagnosis of basaloid squamous cell carcinoma.

White-ivory assay of Drosophila melanogaster under deficient repair conditions.

The prediction ability of a test to detect genotoxic activity may be increased, at least from a theoretical point of view, by carrying it out under deficient repair conditions. The white-ivory (w[i]) assay of Drosophila melanogaster is a somatic mutation and recombination test (SMART) that essentially differs from other SMARTs by the endpoints that can be detected. In this article, we study the consequences, with the w(i) assay, of the introduction of two mutations, mus201 and mei-41, which produce deficiency in two different repair mechanisms: the nucleotide excision repair system and in a G2/M cell-cycle checkpoint, respectively. Ten chemicals, previously classified as positive in the w(i) assay, have been assayed in both deficient repair conditions. As in the w/w+ and mwh/flr3 SMARTs, the results obtained with the w(i) assay show that the use of deficient repair strains does not improve the detection of genotoxic effects. However, the utilization of these deficient repair strains has been shown to be a useful tool in mechanistic studies. In fact, it seems that the nucleotide excision repair system mainly eliminates some spontaneous and chemically-induced damages involved in the reversion of w(i), whereas the repair system deficient in mei-41 flies is partly necessary to recover revertant w(i) spots.

Paranasal sinus fungus balls.

BACKGROUND: Paranasal sinus fungus balls (mycetomas) are a form of fungal sinus infection distinct from allergic fungal sinusitis, fulminant invasive fungal disease, and paranasal aspergillus granulomas. METHODS: The Mayo Clinic surgical pathology files of inflammatory sinus specimens from 1984 to 1994 were examined. Twenty-nine paranasal sinus fungus balls were identified. Cases of allergic fungal sinus and invasive fungal disease were excluded. RESULTS: The fungus ball occurred in 11 men and 18 women, with an age range of 28 to 86 years, mean 64 years. Sinuses involved included maxillary (20 cases), sphenoid (10 cases), ethmoid (9 cases), and frontal (6 cases). In 12 patients, multiple sinuses were involved in a variety of combinations. By culture the most common pathogens were Aspergillus fumigatus and Aspergillus flavus. Treatment was by a variety of surgical procedures. Follow-up in 28 patients showed two recurrences and three deaths due to intracerebral bleed as a complication of surgery. These deaths occurred in patients with sphenoid sinus fungus balls. CONCLUSIONS: Paranasal sinus fungus balls occurs in an elderly population and have a female predominance. They have a low morbidity and recurrence rate. Death can occur in sphenoid sinus lesions as a complication of surgery.

Is the white-ivory assay of Drosophila melanogaster a useful tool in genetic toxicology?

The white-ivory assay of Drosophila is based on the detection of reversions to wild-type phenotype of ommatidia with the white-ivory mutation. A tandem quadruplication of this gene is used in order to increase the reversion probability. Although the exact mechanism implicated in reversion is not known, revertant spots are believed to arise as a consequence of intrachromosmal recombination or related phenomena. Since the white-ivory assay has not been broadly used, the number of chemicals tested until now is still limited. In this work, we have assayed 25 chemicals belonging to several chemical groups, i.e., crosslinking agents, DNA-topoisomerase inhibitors, antimetabolites/nucleotide pool inhibitors, cyclic-adduct inducers, halogenated hydrocarbons, bulky-adduct inducers, intercalating agents, oxidative damage inducers, and a multiple damage inducer, to validate this test. Cross-linking agents, halogenated hydrocarbons, and the multiple damage inducer, dounomycin, were positive. On the contrary, the three antimetabolites/nucleotide pool inhibitors tested were negative. The other chemical groups showed disparate results, since some chemicals were positive, whereas others were negative in each group. A comparison with the results obtained in the w/ w+ and mwh/flr3 assays shows that the wi assay detects a more restricted spectrum of damages than those, although, with respect to carcinogenicity, its sensitivity (0.76, with the 62 chemicals tested until now) is similar to that estimated for the mentioned somatic assays. The conclusion of this work, then, is that the wi assay is not recommended as a general screening test, because the background reversion frequencies show a high variability among solvents, the range of lesion-recognition is lower than in the w/ w+ and mwh/flr3 SMARTs, and the mechanism implicated in the white-ivory reversion is poorly understood.

Columnar cell carcinoma of the thyroid: report of three additional cases.

Columnar cell carcinoma is a recently described variant of thyroid carcinoma that has been associated with an aggressive clinical course. The authors describe three new cases of columnar cell carcinoma occurring in two women and one man aged 62, 46, and 46 years, respectively. The tumors ranged in size from 1 to 7.5 cm, and two of the tumors were associated with distant metastases. One patient died of disease 39 months after presentation. Another patient is alive with distant metastases 27 months after diagnosis. One patient appears to be a long-term survivor with no evidence of metastasis after follow-up of 22 years. This patient had a tumor that was small (1 cm) and encapsulated. DNA ploidy analysis in two tumors showed diploid DNA content, and there was no elevated S phase. All tumors were positive for thyroglobulin and negative for calcitonin and carcinoembryonic antigen (CEA). These findings support the original observation that columnar cell variants of papillary thyroid carcinoma are usually aggressive neoplasms. There does not appear to be an increased incidence of DNA aneuploidy in columnar cell carcinomas to account for their more aggressive behavior. These tumors occur over a wide age range, can metastasize widely, and are not usually responsive to radioactive iodine or chemotherapy.

Somatic recombination, gene amplification and cancer.

The principle objective of this research programme, to analyse chemical induction of somatic recombination and related endpoints, i.e., mobilization of transposing elements and gene amplification, has been approached by means of several assay systems. These have included Drosophila, Saccharomyces and mammalian cell cultures. 6.1. Screening assays for mitotic recombination. A large number of chemicals have been investigated in the three Drosophila assay systems employed--the multiple wing hair/flare wing spot system developed by Graf et al., 1984, the white-ivory system developed by Green et al., 1986 and the white/white+ eye spot assay developed by Vogel (Vogel and Nivard, 1993). Particularly the screening of 181 chemicals, covering a wide array of chemical classes, by the last mentioned assay has shown that measurement of somatic recombination in Drosophila constitutes a sensitive and efficient short-term test which shows a remarkably good correlation with the agent score of 83 short-term tests analysed by ICPEMC (Mendelsohn et al., 1992; Table 2) as well as the assay performance in international collaborative programmes measuring carcinogen/non-carcinogens (de Serres and Ashby, 1981; Ashby et al., 1985, 1988). Also the wing spot assay has gained wide international recognition as a similarly sensitive test. These two assay systems in Drosophila measure both intrachromosomal events and interchromosomal recombination. The white-ivory system on the other hand is based on the loss of a tandem duplication in the white locus, the mechanism of which is less known, but probably involves intrachromosomal recombination. The difference in the mechanism between this assay and the former two was indicated by the lack of response to methotrexate in the white-ivory assay, while this compound was strongly recombinogenic in both the wing spot and white/white+ assays. The use of different strains of Drosophila with the white/white+ assay demonstrated the importance of the background genotype for the outcome of the test. Up to a 60-fold variation was found between the different genotypes in the response to procarcinogens, evidently dependent on differences in the metabolic activation of procarcinogens. In 1989 Schiestl presented results on intrachromosomal recombination in the strain RS112 of Saccharomyces, which indicated a capability to detect a range of chemical carcinogens, which gave negative results in Ames Salmonella assay. Such a test system, which could identify a larger range of potential carcinogens than conventional short-term tests evidently would be of great value and it therefore seemed of importance to follow up the observations by Schiestl. However, studies within this programme on the same strain of Saccharomyces, as well as the strains D7 (measuring intragenic recombination, intergenic recombination, and point mutation) and JD1 (measuring intragenic recombination at two loci) could not support the observations and interpretation by Schiestl (1989). The Drosophila white-ivory system, which presumably responds primarily by intrachromosomal recombination, did not give positive results with these Salmonella-negative agents either. One system to measure mitotic recombination in mammalian cell cultures was developed in the present programme. It was based on heterozygous mutations in both alleles of the adenosine deaminase gene (ADA). The system selects for proficient recombinants generated by the deficient cells. So far only pilot experiments, indicating that this experimental system operates as planned, have been performed. 6.2 Mechanisms of mitotic recombination The induction of mosaic spots in the wing spot and the white/white+ assays is predominantly dependent on interchromosomal recombination. This is evident from the fact that heterozygous inversions reduce the frequency of spots. A relationship between the length of inversions and the reduction of spots was demonstrated in the white/white+ assay--the long inversion ln(l)sc4L

The epithelioid blue nevus. A multicentric familial tumor with important associations, including cardiac myxoma and psammomatous melanotic schwannoma.

We report on 21 pigmented nevi that occurred in 11 patients (6 male and 5 female patients) age 3 to 39 years. All patients had the Carney complex (myxomas, spotty skin pigmentation, endocrine overactivity, and schwannomas); six patients were members of three families. The nevi occurred on the extremities and trunk, less frequently on the head and neck, and were multiple in five patients. Clinically, they were darkly pigmented, domed, and small (less than 1 cm) and commonly interpreted as "blue nevi." Microscopically, they featured heavily pigmented, poorly circumscribed, dermal lesions that displayed two types of melanocytes: one intensely pigmented, globular, and fusiform; the other lightly pigmented, polygonal, and spindle. The melanocytes were situated among the dermal collagen bundles singly, in short rows and small groups, and occasionally in fascicles. Nuclei of the lightly pigmented, polygonal and spindle cells were vesicular with very pale chromatin and a single prominent nucleolus. Seven tumors were each part of a combined nevus. After excision, none of the tumors recurred or metastasized. The epithelioid blue nevus is important because of its strong association with the Carney complex. Therefore, affected patients (and their relatives) should be considered at risk for other elements of the syndrome, especially cardiac myxoma.

Solitary fibrous tumour of the major salivary glands.

We report four cases of solitary fibrous tumours which involved the parotid (two cases), submandibular, and sublingual glands of two men and two women ranging in age from 46 to 81, mean 66 years. The tumours presented as painless, firm masses which involved the substance of the salivary gland in each case. All have had an uneventful clinical course after local excision. Awareness of this entity is important to avoid confusion with haemangiopericytoma.

'Non-genotoxic' carcinogens evaluated using the white-ivory assay of Drosophila melanogaster.

Seven carcinogenic compounds (urethane, ethionine, auramine O, safrole, amitrole, acetamide and thioacetamide) were tested using the white-ivory (Wi) assay of Drosophila melanogaster. These compounds were chosen because they were considered as Ames-test negative but produced positive results in the yeast DEL assay, which estimates the introduction of intrachromosomal recombination. Only one compound, urethane, produced positive results in the Wi assay, while the remaining were classified as negative. These results indicate that, in contrast with which has been postulated in yeast, these carcinogens do not induce any event associated to intrachromosomal recombination in D. melanogaster.

Salivary duct carcinoma. Clinicopathologic and immunohistochemical review of 26 cases.

BACKGROUND: Salivary duct carcinoma (SDC) is a high grade aggressive malignancy of the major salivary glands. Clinical and pathologic features that may be predictive of survival are not well delineated. The microscopic features of SDC are remarkably similar to those of mammary ductal carcinoma, raising the question of whether these tumors share antigenic or hormonal features. METHODS: We reviewed the clinical and pathologic characteristics of 26 cases of SDC treated at the Mayo Clinic from 1960 to 1989. Immunoperoxidase studies and flow cytometry were performed in 25 and 24 cases, respectively. RESULTS: The study population consisted of 22 men and 4 women (mean age, 66 years). The parotid gland was involved in 23 patients and the submaxillary gland in 3. Five of 24 tumors studied were diploid (21%), and 19 (79%) were nondiploid. Nine tumors (35%) recurred locally and 16 (62%) metastasized distantly; 20 patients (77%) died of disease at a mean interval of 3 years after diagnosis. Female sex was the only significant negative prognostic factor analyzed, but positive nodal status approached significance. Paraffin-section immunostaining showed positive reactions for epithelial membrane antigen (100%), keratin (AE1/AE3) (88%), alpha-lactalbumin (88%), GCDFP-15 (76%), and carcinoembryonic antigen (72%); S-100 protein was rarely detected (4%). Stains for estrogen receptor were uniformly negative, but one tumor was positive for progesterone receptors. CONCLUSIONS: The prognosis for SDC is dismal, and clinically useful prognostic factors were not found. Our results do not confirm hormonal concordance between SDC and breast carcinoma.

Accuracy of frozen section diagnosis in surgical pathology: review of a 1-year experience with 24,880 cases at Mayo Clinic Rochester.

OBJECTIVE: To determine the accuracy of frozen section examination for routine diagnostic use in surgical pathology. DESIGN: We retrospectively reviewed the experience with frozen sections at Mayo Clinic Rochester during calendar year 1993. MATERIAL AND METHODS: The results in 24,880 cases (97,914 frozen section slides) processed during 1993 were compared with findings on permanent sections, and types of errors detected were classified into one of three quality assurance categories. RESULTS: The overall rate of frozen section accuracy was 97.8%. Of the 2.2% of surgical pathology reports that needed revision, 1.6% were unavoidable tissue sampling errors, 0.5% were errors that reflected change in degree of abnormality, and only 0.1% were clinically significant errors that may have affected patient management. CONCLUSION: Frozen section diagnosis is accurate for processing a high volume of surgical pathology cases.

Accuracy of frozen-section diagnosis of mammographically directed breast biopsies. Results of 1,490 consecutive cases.

The accuracy of intraoperative frozen-section diagnosis of small in situ and invasive breast cancers is uncertain. We reviewed the results of 1,490 consecutive wire-localized breast biopsies from 1,439 patients with nonpalpable mammographically detected abnormalities examined by frozen section at Mayo Clinic over a 3-year period. The mammographic abnormalities included benign calcifications (61 cases), indeterminate calcifications (422 cases), worrisome calcifications (54 cases), well-circumscribed nodules (115 cases), irregular nodules (473 cases), architectural distortions (52 cases), asymmetry (39 cases), well-circumscribed nodules with calcification (12 cases), irregular nodules with calcification (75 cases), architectural distortions with calcification (35 cases), and asymmetry with calcifications (12 cases). We detected 457 carcinomas, including 135 in situ and 322 invasive cancers. The invasive carcinomas had a mean diameter of 1.07 cm (range, 0.1-3.0 cm), including 191 with diameters of 1 cm or less (59.3% of cases). In 77 cases (5.2% of total), the diagnosis was deferred to permanent sections. Frozen-section accuracy was 97.7%. False-negative diagnoses were rendered in 0.5% of cases, and there were no false-positive diagnoses. There was no correlation of infiltrating carcinoma diameter and error rate. These results indicate that intraoperative frozen section of mammographically directed breast biopsies provides accurate and reliable diagnoses.

Methodological aspects of the white-ivory assay of Drosophila melanogaster.

The white-ivory somatic assay of Drosophila melanogaster was developed to detect genotoxic agents which induce loss of a tandem duplication. Although the mechanism of this loss is not known, some suggestions point to intrachromosomal recombination as the main reversion mechanism. Since the few papers published to date on this assay present controversial methodologies, prior to a larger study of chemicals with different mechanisms of action, we have carried out an analysis to optimize some conditions of this assay. For this purpose, we have used three different strains and four well characterized mutagenic chemicals: N-ethyl-N-nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and hexamethyl phosphoramide (HMPA). The results obtained allow us to conclude that: (i) the best strain for this assay is C(1)DX,y,f/Dp(1:1:1:1)wi,y2, although the use of strain FM6,l(1)66a/Dp(1:1:1:1)wi,y2;st/st could be considered for some mechanistical studies; (ii) developmental reasons make it necessary to use as estimate of reversion frequency the proportion of eyes showing at least one spot; (iii) reversion frequency cannot be used as estimate of mutation efficiency, neither can spot size evaluate time of spot induction; (iv) the four chemicals clearly induce loss of the wi duplication; according to their activities they rank ENU > HMPA > MMS approximately EMS.

Hyaline-cell rich chondroid syringoma. A tumor mimicking malignancy.

We report three cases of chondroid syringoma composed almost entirely of hyaline cells and occurring in the soft tissues. These lesions were confused with a variety of malignant neoplasms, including malignant melanoma, myxoid chondrosarcoma, and alveolar soft part sarcoma. The tumors occurred in two men and one woman aged 37, 75, and 71 years, respectively, and involved the heel, great toe, and face. All cases arose in the subcutaneous tissue or fascial layer and ranged in size from 0.8 to 2.0 cm. The neoplasms were composed of plasmacytoid hyaline cells with variable amounts of myxoid stroma. Two of the chondroid syringomas contained rare duct structures. One contained cartilage lobules but no ducts. All were well circumscribed. Immunohistochemistry showed positive staining for keratin, S-100, and vimentin in all cases. Muscle markers, including desmin, smooth-muscle actin, and muscle-specific actin, were negative. Follow-up has showed no evidence of recurrence or metastasis.

Oncocytic differentiation in salivary gland tumours.

An oncocytic mucoepidermoid carcinoma and an oncocytic pleomorphic adenoma occurred in a 47-year-old male and a 75-year-old female, respectively. Both presented as asymptomatic parotid gland masses without evidence of facial nerve paralysis and were treated by superficial parotidectomy. There has been no evidence of recurrence or metastasis. Oncocytic change is rare in major salivary gland mucoepidermoid carcinoma with only two previously reported cases. Marked oncocytic transformation of pleomorphic adenomas can cause their confusion with oncocytomas. Recognition of oncocytic differentiation in various salivary gland tumours is important to avoid misclassification of these lesions.

Immunohistochemical analysis of salivary gland canalicular adenoma.

Canalicular adenoma is a newly recognized salivary gland adenoma that may be confused with malignant salivary gland tumors. To better characterize this neoplasm, six examples were investigated with a panel of immunohistochemistry antibodies including anti-keratin (AE1/AE3), anti-epithelial membrane antigen, anti-carcinoembryonic antigen, anti-vimentin, anti-S-100, anti-muscle specific actin, and anti-glial fibrillary acid protein. All canalicular adenomas stained in a similar fashion showing positive staining with anti-keratin, anti-vimentin, and anti-S-100 (6 of 6 cases each). Rare focal staining with anti-epithelial membrane antigen and anti-glial fibrillary acid protein was noted (1 of 6 cases each). This immunohistochemistry staining pattern was compared with those of ameloblastoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma. Immunohistochemistry may be useful in the distinction of canalicular adenoma from other salivary gland tumors.